1. High yield purification of Helicobacter pylori neutrophil-activating protein overexpressed in Escherichia coli
- Author
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Chung-Chu Chen, Zhi-Wei Hong, Te-Lung Tsai, Hua-Wen Fu, Han-Wen Chang, Yu-Chi Yang, and Ting-Yu Kuo
- Subjects
Batch chromatography ,Lysis ,Sephadex ,medicine.disease_cause ,law.invention ,Bacterial Proteins ,law ,mental disorders ,Native state ,medicine ,Escherichia coli ,Protein secondary structure ,DEAE ,chemistry.chemical_classification ,Reactive oxygen species ,Chromatography ,biology ,Helicobacter pylori ,Negative chromatography ,pH ,musculoskeletal, neural, and ocular physiology ,fungi ,E. coli ,ROS ,Hydrogen-Ion Concentration ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,HP-NAP ,Biochemistry ,chemistry ,Solubility ,Recombinant DNA ,human activities ,psychological phenomena and processes ,Biotechnology ,Research Article - Abstract
Background Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-induced gastric inflammation. Due to its immunogenic and immunomodulatory properties, HP-NAP has been used for developing vaccines against H. pylori infection and new drugs for cancer therapy. Results Here, we provide a simple process for high-yield production of HP-NAP by applying one-step negative chromatography to purify recombinant HP-NAP expressed in Escherichia coli (E. coli). In our E. coli expression system, recombinant HP-NAP constitutes nearly 70% of the total protein. Overexpressed recombinant HP-NAP is almost completely soluble upon cell lysis at pH 9.5. Under the optimal condition at pH 8.0, recombinant HP-NAP with purity higher than 95% can be obtained from E. coli by collecting the unbound fraction using diethylaminoethyl (DEAE) Sephadex resin in batch mode. The overall yield of HP-NAP from a 50-ml E. coli culture is ~19 mg. The purified HP-NAP folds into a multimer with a secondary structure of α-helix and is able to trigger the production of reactive oxygen species by neutrophils. Conclusions Purification of recombinant HP-NAP overexpressed in E. coli using DEAE Sephadex negative mode batch chromatography is an efficient method for high-yield production of highly pure HP-NAP in its native state. The purified HP-NAP is useful for various clinical applications including vaccine development, diagnosis, and new drug development.
- Published
- 2015